Molecular Analysis of Transferable Tetracycline Resistance Plasmids from Clostridium perfringens

Transcription

1 JOURNAL OF BACTERIOLOGY, Feb. 1985, p /85/2636-5$2./ Copyright 1985, American Society for Microbiology Vol. 161, No. 2 Molecular Analysis of Transferable Tetracycline Resistance Plasmids from Clostridium perfringens LAWRENCE J. ABRAHAM* AND JULIAN I. ROOD Division of Veterinary Biology, School of Veterinary Studies, Murdoch University, Murdoch, Western Australia 615, Australia Received 21 March 1984/Accepted 9 October 1984 Conjugative tetracycline resistance plasmids from 15 Clostridium perfringens isolates from piggeries were analyzed by restriction endonuclease digestion and agarose gel electrophoresis. Seven isolates from one farm were found to carry a 47-kilobase pair (kb) plasmid, pjir5, which had EcoRI, XbaI, and ClaI profiles that were identical to those of a previously characterized plasmid, pcw3. An isolate from a second farm was found to carry a plasmid, pjir6, which also was indistinguishable from pcw3. Five additional isolates from a third farm carried a 67-kb plasmid, pjir2, which had at least 29 kb of DNA in common with pcw3. Finally, two isolates from a fourth farm were found to carry a 5-kb plasmid pjir4, which appeared to consist of an entire pcw3 molecule with a 3-kb insertion. Comparative restriction maps of pcw3, pjir2, and pjir4 that identified the regions of homology among these plasmids were constructed. We suggest that many conjugative tetracycline resistance plasmids in C. perfiringens may contain a pcw3-like core. Clostridium perfringens is an anaerobe that forms a part of the normal intestinal flora of humans and animals. This organism is the causative agent of such diseases as gas gangrene in humans, hemorrhagic enteritis of calves, and enterotoxemia of sheep. It is also a secondary pathogen in various disease conditions, such as necrotic enteritis of chickens (6). In recent years there has been renewed interest in the clostridia as they have been implicated in a number of diseases in humans. For instance, Clostridium difficile is known to cause antibiotic-associated pseudomembranous colitis (4). Consequently, there is a need to understand the genetic mechanisms whereby these anaerobes can interact, especially with regard to the transfer of drug resistance determinants. Resistance transfer was first demonstrated in C. perfringens by Sebald and Brdfort (12). These workers identified a conjugative R-plasmid, pip41, which carries the tetracycline and chloramphenicol resistance genes (1). Subsequently, Rood et al. (11) characterized a C. perfringens strain (strain CW92) that was isolated from a specimen of human origin in California. This strain was chloramphenicol and tetracycline resistant, but was able to transfer only its tetracycline resistance. A conjugative tetracycline resistance plasmid was identified in strain CW92 and designated pcw3 (11). Rood et al. (1) isolated many multiply antibiotic-resistant strains of C. perfringens from porcine feces that were obtained from several farms in Wisconsin. Recently, Rood (8) reported that a significant proportion of the antibiotic-resistant strains obtained in that study have the ability to transfer their tetracycline resistance to appropriate recipient strains. In this paper we describe the plasmids responsible for this property and report studies on the comparative molecular analysis of these plasmids. (Some of the results have been reported previously [Abraham and Rood, Aust. Microbiol. 4:85, 1983].) * Corresponding author. 636 MATERIALS AND METHODS Bacterial strains, media, and chemicals. Plasmid-free strains CW54 and JIR39 are derivatives of strain CW362 (7, 8). Wild-type strains were isolated from porcine and human feces obtained from four farms in Wisconsin (1). All other strains were tetracycline-resistant transconjugants of strain CW54 or JIR39. All strains were maintained and propagated as described previously (8). A list of the plasmids identified during this study is presented in Table 1. Nutrient agar and Trypticase-peptone-glucose broth were prepared and used as described previously (1). Tetracycline was a generous gift from Upjohn Pty. Ltd., Sydney, Australia. Growth of cells and isolation of plasmid DNA. Cells were cultured overnight on nutrient agar containing 5,ug of tetracycline per ml and then subcultured in 1 ml of Trypticase-peptone-glucose broth. Mid-log phase cells were washed and suspended in 2 ml of 25% sucrose in 3 mm TES (Tris-hydrochloride buffer [ph 8.] containing 5 mm EDTA and 5 mm NaCl). After treatment with.4 ml of a lysozyme solution (1 mg/ml in TES) for 15 min at 37 C and with.8 ml of 25 mm EDTA (ph 8.) for an additional 5 min, lysis was achieved by adding.5 ml of 1% (wt/vol) sodium dodecyl sulfate and incubating the preparation at 37 C for 5 min. The lysate was then treated with NaCl by the method of Guerry et al. (2). The resultant lysates were subjected to cesium chloride-ethidium bromide density gradient centrifugation for 2 h at 49, rpm (type 7.1Ti rotor; Beckman model L-8 ultracentrifuge). Plasmid bands were extracted through the top of the tube, and ethidium bromide was extracted with NaCl-saturated propan-2-ol. The preparations were dialyzed against.1 x TES, concentrated by pervaporation, and then dialyzed again before storage at 4 C. Restriction endonuclease digestion and agarose gel electrophoresis. All restriction endonucleases were obtained from Boehringer-Mannheim or Bethesda Research Laboratories and were used according to the instructions of the suppliers. Electrophoresis of DNA samples was carried out in.6 to 1.2% agarose in a horizontal gel apparatus by using a Tris-acetate electrode buffer system, as described by

2 VOL. 161, 1985 C. PERFRINGENS TETRACYCLINE RESISTANCE PLASMIDS 637 TABLE 1. Plasmids and their origins Plasmid Source Size (kb) Phenotype pcw3 Strain CW92' 47 Tra+ TCrb pjir1 Farm A (porcine) ca. 12 Cryptic pjir2 Farm G (porcine) 67 Tra+ Tcr pjir3 Farm A (human) ca. 4.5 Cryptic pjir4 Farm I (porcine) 5 Tra+ Tcr pjir5 Farm A (porcine and human) 47 Tra+ Tcr pjir6 Farm H (porcine) 47 Tra+ Tcr a See reference 11. btra+, Conjugation; Tcr, tetracycline resistance. ' Farm designations are explained in reference 1. Young et al. (16). The sizes of DNA fragments were estimated by comparing their mobilities with those of HindIIIcleaved XcI857, using a computer program (9). DNA hybridization studies. DNA fragments in agarose gels were denatured and transferred to nitrocellulose filters (15). The filters were baked for 2 h at 8 C under a vacuum. DNA derived from pcw3 was labeled by nick translation with 32P-labeled dctp (Amersham Corp.). Heat-denatured probe DNA was then hybridized to the DNA present on the nitrocellulose filters as described previously (14). Hybridized probe DNA was detected by autoradiography, using intensifying screens. RESULTS Examination of wild-type plasmid profiles. Previous studies (8) showed that 15 of the 89 tetracycline-resistant wild-type C. perfringens strains that were available from the original Wisconsin study (1) could transfer their antibiotic resistance to suitable recipient strains. In the present study, plasmid DNA was prepared from each of the 15 wild-type isolates, and the plasmids were examined by agarose gel electrophoresis. The profiles that were obtained could be correlated readily with the farm of origin (data not shown). The common feature of all 15 profiles was the presence of at least one high-molecular-weight plasmid. However, since many different low-molecular-weight plasmids also were present, it was not possible to make any absolute correlations between the transferable tetracycline resistance phenotype and the presence of a specific plasmid. Therefore, we decided to examine the transconjugants that were available from the previous study (8). Examination of transconjugants. For each wild-type donor, four independently derived transconjugants were examined initially; three of these were from interstrain matings of the wild type with strain CW54, and one was from an intrastrain mating of a strain CW54-derived transconjugant with strain JIR39 (8). Agarose gel electrophoresis indicated that a high-molecular-weight plasmid was present in all 6 of the transconjugants that were examined (data not shown). Four strain CW54-derived transconjugants also were found to contain an additional low-molecular-weight plasmid. One of these transconjugants, which was from a farm A porcine isolate, harbored a plasmid (pjir1; ca. 12 kilobase pairs [kb]) that was present in all of the farm A wild types. Although this transconjugant could in turn donate its tetracycline resistance, plasmid pjir1 was not detected in the strain JIR39 derivative that was produced. The remaining three strain CW54-derived transconjugants that also carried a low-molecular-weight plasmid were all derived from matings with a single human farm A isolate. This plasmid (pjir3; ca. 4.5 kb) also was present in the wild-type parent. Similarly, when one of these strains donated its tetracycline resistance, plasmid pjir3 was not detected in the resulting strain JIR39-derived transconjugant. Because of the absence of any phenotypic markers on pjir1 or pjir3, it was not possible to determine whether these plasmids were mobilized by other plasmids present in the wild-type isolates or whether these small plasmids were self-transmissible. Considering the small sizes of pjir1 and pjir3, it is unlikely that they encode enough information to mediate their own transfer. A wider, more systematic study will be required to establish whether the high-molecular-weight plasmids detected in this study have the ability to mobilize other nonconjugative plasmids present within the same host. Restriction analysis of plasmids. To further characterize the plasmids present in each of the 6 transconjugants, a restriction analysis was carried out. We found that within each group of four transconjugants, the same restriction profiles were obtained. We concluded that the same highmolecular-weight plasmid was present in all four transconjugants within each group. Consequently, further studies were restricted to one transconjugant derived from each of the 15 wild-type donors. Our results also showed that all seven transconjugants from farm A donors carried plasmids with identical restriction profiles. These plasmids were all designated pjir5. An additional plasmid, which was derived from the single farm kbi k b- 2.1 kb- 1.9 kb FIG. 1. Agarose gel electrophoresis of EcoRI-digested plasmid DNA derived from transconjugants. Plasmid DNA was digested with EcoRI, and the resulting fragments were subjected to electrophoresis in a.8% (wt/vol) agarose gel at 75 V for 5 h. The sizes of the pcw3 fragments are indicated. The lane labeled lambda contains fragments of known size produced when bacteriophage XcI857 was digested with HindIll.

3 638 ABRAHAM AND ROOD r 2 v " coa (C) 4~ a a a QI 7 kb FIG. 2. Agarose gel electrophoresis of XbaI-digested plasmid DNA derived from transconjugants. Plasmid DNA was digested with XbaI, and the resulting fragments were subjected to electrophoresis in a.8% (wt/vol) agarose gel at 35 V for 16 h. The gel was then treated as described in the legend to Fig. 1. The sizes of the HindIlI-digested XcI857 fragments are indicated. H isolate, was designated pjir6. Addition of the sizes of the EcoRI and ClaI fragments established a molecular size of 47 kb for pjir5 and pjir6. The EcoRI (Fig. 1), XbaI (Fig. 2), and ClaI (data not shown) profiles of pjir5 were compared with the restriction profiles of a previously characterized tetracycline resistance plasmid, pcw3 (11). pjir5, pjir6, and pcw3 had identical restriction profiles for all three enzymes. The five transconjugants from farm G isolates also carried plasmids with a common restriction profile. The 67-kb plasmid in these transconjugants was designated pjir2. A comparison of the restriction profiles of pjir2 and pcw3 indicated that pjir2 was different from pcw3, although both plasmids yielded some fragments of the same size. An examination of the EcoRI digests of pjir2 and pcw3 (Fig. 1) showed that six EcoRI fragments (8.3, 3.8, 3.8, 2.5, 2.1, and 1.9 kb) were shared by both of these plasmids. XbaI digestion of pjir2 and pcw3 (Fig. 2) confirmed that pjir2 was different than pcw3 and showed that eight fragments with a total size of 14.5 kb were common to both plasmids. A comparison of the ClaI profiles of pjir2 and pcw3 confirmed these results. The two transconjugants from the farm I isolates had plasmids with identical restriction patterns. These 5-kb plasmids were designated pjir4. A comparison of their restriction profiles showed that pjir4 was very similar to pcw3. The EcoRI-generated fragments of pjir4 were identical to those of pcw3, except that one of the 3.8-kb fragments present in pcw3 was absent and three new fragments (3.5, 2.2, and.6 kb) were present in pjir4 (Fig. 1). The XbaI-generated fragments of pjir4 were the same as those of pcw3, except that pjir4 had a 3.1-kb fragment that was not present in the equivalent pcw3 digest (Fig. 2). The ClaI results confirmed that pjir4 was very similar to pcw3. DNA hybridization studies were used to confirm the results. Plasmid DNA derived from pcw3 was nick translated by using 32P-labeled dctp and hybridized to EcoRIdigested pcw3, pjir2, and pjir4. The results showed that all of the EcoRI fragments that were present in both pcw3 and pjir4 or in both pcw3 and pjir2 hybridized with DNA derived from pcw3 (Fig. 3). Comparative analysis of pjir2, pjir4, and pcw3. As pjir2 and pjir4 were found to be different from pcw3, we decided to define the relationship among these three plasmids. A complete restriction map of pcw3 was generated (Abraham and Rood, manuscript in preparation), and then partial restriction maps of pjir2 and pjir4 were deduced by comparing restriction digests of these two plasmids with equivalent pcw3 digests. In this way we were able to map the regions of pjir2 and pjir4 that were the same as those in pcw3. Plasmid pjir4 was missing the 11.7-kb ClaI fragment of pcw3 that extended from 28.4 to 4.1 kb on the linear pcw3 map (Fig. 4), but a new 14.5-kb ClaI fragment was present. The EcoRI profiles of pjir4 and pcw3 were identical, except that one of the 3.8-kb fragments of pcw3 was missing in pjir4. Both of these fragments are within the 11.7-kb ClaI fragment. By carrying out EcoRI-HpaI double digestions (data not shown), it was possible to identify which of the two fragments was absent in pjir4 and thus identify kb kbw kb kb 2.5 kb 2.1 kb kbm 1.2 kb- " '"' J. BACTERIOL. FIG. 3. Southern blot analysis of pjir2 and pjir4. Hybridization and Southern blot analysis were carried out as described in the text. DNA derived from pcw3 was labeled with 32P and used to probe EcoRI-digested plasmid DNAs from pcw3, pjir2, and pjir4. The sizes of the pcw3-derived EcoRI fragments are indicated.

4 VOL. 161, 1985 C. PERFRINGENS TETRACYCLINE RESISTANCE PLASMIDS 639 pcw3 47 kb - i -- -i A o. out- a U '1A,II St i al -.. I 1 - -O o 9 4 * le v Mt q I -.. a: ' -om: 19 1 ' 11: ----, OC- OZO O O O a O A,; _ O a - a s a X IT I I. -I Xi7 I I-I pjir2 67 kb -X o) - A o o. C6 C 4. XL u, uu-qu c f X,,T I 'I I --. a g C, Z. I 'ST Y 7 o M t u I I T C - y ~I'i~ A I I AUX.,,, _ u _ ā- -_,. g,x,,, P. a q U -Q U u X. Ui fa tn Li EA pjir4 II I1'IV I Mt y I I vr I I W I I I 5kb FIG. 4. Linear restriction maps of pcw3, pjir2, and pjir4. The complete map of pcw3 is linearized at the unique SphI site. The regions shown on the pjir2 and pjir4 maps are those parts common to pjir2 or pjir4 and pcw3. the region of pjir4 that was different from pcw3. The results showed that the first 3.8-kb EcoRI fragment of pcw3 (29.6 to 33.4 kb) (Fig. 4) was replaced by 3.5-, 2.2-, and.6-kb EcoRI fragments in pjir4, which accounts for the 3-kb increase in size of this plasmid compared with pcw3. Comparative restriction data also indicated that pjir2 shared a substantial region of homology with pcw3. Delineation of the regions common to pjir2 and pcw3 was achieved by comparing double digests, using ClaI and one of a variety of other restriction enzymes. From these studies we concluded that pjir2 contains two regions that are not homologous with pcw3. The location of these regions is indicated in Fig. 4. DISCUSSION The study reported by Rood (8) was the first in which a large number of wild-type antibiotic-resistant strains of C. perfringens were examined for their ability to transfer their resistance. He showed that 15 of the 89 tetracycline-resistant isolates from the Wisconsin porcine study (1) donated their tetracycline resistance to recipient strains. The results presented here show that a plasmid molecule was present in all of the tetracycline-resistant strain CW54-derived transconjugants that were from the 15 wild-type isolates. An analysis of plasmids from the strain JIR39-derived transconjugants demonstrated that the plasmids were stable on subsequent transfer. From the results we concluded that the plasmids involved in transfer carried the genes responsible for both conjugal transfer and tetracycline resistance. Restriction analysis has frequently been employed to examine the molecular relationships among various R-plasmids (3, 13). These studies are often carried out to determine whether R-plasmids from different sources are related or whether they have a common evolutionary origin. This report represents the first study in which this approach has been used for the analysis of conjugative R-plasmids from C. perfringens. The most significant finding from these experiments is that conjugative R-plasmids isolated from C. perfringens strains of porcine origin in Wisconsin appear to be the same as pcw3, which was found in a human hospital isolate from California. Although these pcw3-like plasmids all have the same restriction profiles, small DNA sequence differences cannot be excluded, and so each plasmid was named individually. The fact that pjir6 was found in the conjugative tetracycline-resistant isolate from farm H and the fact that pjir5 was found in all of the conjugative tetracyclineresistant isolates from farm A, regardless of whether the isolate was of porcine or human origin, suggest that there is free genetic exchange between porcine and human C. perfringens strains. This conclusion is supported by the apparent identity of both pjir5 and pjir6 with the human hospital plasmid, pcw3. It is also significant that pjir2 and pjir4 showed substantial homology with pcw3. In fact, pjir2 appears to consist of a pcw3-like plasmid with two non-homologous additional regions of at most 38 kb of DNA. The regions of nonhomology are smaller than indicated, as we have not determined exactly which segments of the individual restriction fragments that are unique to pjir2, are the same as in pcw3. Plasmid pjir4 also was found to be similar to pcw3. Restriction analysis revealed that pjir4 is a pcw3-like plasmid with only 3 kb of additional DNA that is unique to pjir4. Although we have not mapped the precise location of the additional DNA, restriction analysis indicates that it consists of a single insertion of 3 kb of DNA into a 3.8-kb EcoRI fragment of pcw3. These findings have significant implications for our understanding of the molecular evolution of transferable tetracycline resistance in C. perfringens. The ubiquity of a pcw3- like element in transferable tetracycline-resistant strains in this study raises the possibility that pcw3 or a pcw3-like plasmid forms the basis of all transferable R-plasmids in C. perfringens. It appears that this pcw3-like core is able to acquire additional DNA (as with pjir2 and pjir4) by a mechanism that is as yet undetermined and is able to transfer this DNA to other members of the population by a conjugation-like process. It would be of great interest to study the regions surrounding the sites of heterologous insertion within pjir2 and pjir4 to possibly determine how this DNA was inserted. Although transposable elements have not been demonstrated in this species. they have been shown to be involved in the acquisition of additional genetic information by plasmids in other species (5). Current studies in our laboratory are aimed at the characterization of transferable drug resistance plasmids from a wider geographical range to establish whether the pcw3-like plasmid is truly ubiquitous. ACKNOWLEDGMENTS This work was supported by grants from the Australian Research Grants Scheme and Murdoch University. L.J.A. is the recipient of a Commonwealth Postgraduate Research Award. We are pleased to acknowledge Lyn M. Rowland for meticulous and cheerful technical assistance. Thanks are also extended to J. Shine for a generous gift of restriction enzymes. LITERATURE CITED 1. Brefort, G., M. Magot, H. lonesco, and M. Sebald Characterization and transferability of Clostridium perfringens plasmids. Plasmid. 1:52-66

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